Recent advances in molecular and genetic techniques have led to establishment of new biomedical fields; however, morphological techniques are still required for a more precise understanding of functioning cells and tissues. Conventional preparation procedures involve a series of chemical fixation, alcohol dehydration, paraffin or epoxy resin embedding, sectioning, and staining steps. In these steps, technical artifacts modify original morphologies of the cells being examined. Furthermore, difficulties are associated with capturing dynamic images in vivo using conventional chemical fixation. Therefore, a quick-freezing (QF) method was introduced for biological specimens in the 20th century. However, specimens have to be resected from living animal organs with blood supply, and their dynamical morphologies have not been investigated in detail using the QF method. In order to overcome these issues, the tissue resection step of organs had to be avoided and samples needed to be frozen under blood circulation. Our in vivo cryotechnique (IVCT) was an original technique to cryofix samples without resecting their tissues. The most significant merit of IVCT is that blood circulation into organs is preserved at the exact moment of freezing, which has been useful for arresting transient physiological processes of cells and tissues and maintaining their components in situ.

In chronic inflammatory lesions of autoimmune and infectious diseases, plasma cells are frequently observed. Antigens recognized by antibodies produced by the plasma cells mostly remain unclear. A new technique identifying these corresponding antigens may give us a breakthrough for understanding the disease from a pathophysiological viewpoint, simply because the immunocytes are seen within the lesion. We have developed an enzyme-labeled antigen method for microscopic identification of the antigen recognized by specific antibodies locally produced in plasma cells in inflammatory lesions. Firstly, target biotinylated antigens were constructed by the wheat germ cell-free protein synthesis system or through chemical biotinylation. Next, proteins reactive to antibodies in tissue extracts were screened and antibody titers were evaluated by the AlphaScreen method. Finally, with the enzyme-labeled antigen method using the biotinylated antigens as probes, plasma cells producing specific antibodies were microscopically localized in fixed frozen sections. Our novel approach visualized tissue plasma cells that produced 1) autoantibodies in rheumatoid arthritis, 2) antibodies against major antigens of Porphyromonas gingivalis in periodontitis or radicular cyst, and 3) antibodies against a carbohydrate antigen, Strep A, of Streptococcus pyogenes in recurrent tonsillitis. Evaluation of local specific antibody responses expectedly contributes to clarifying previously unknown processes in inflammatory disorders.

Several theories have been proposed regarding pain transmission mechanisms in tooth. However, the exact signaling mechanism from odontoblasts to pulp nerves remains to be clarified. Recently, ATP-associated pain transmission has been reported, but it is unclear whether ATP is involved in tooth pain transmission. In the present study, we focused on the vesicular nucleotide transporter (VNUT), a transporter of ATP into vesicles, and examined whether VNUT was involved in ATP release from odontoblasts. We examined the expression of VNUT in rat pulp by RT-PCR and immunostaining. ATP release from cultured odontoblast-like cells with heat stimulation was evaluated using ATP luciferase methods. VNUT was expressed in pulp tissue, and the distribution of VNUT-immunopositive vesicles was confirmed in odontoblasts. In odontoblasts, some VNUT-immunopositive vesicles were colocalized with membrane fusion proteins. Additionally P2X3, an ATP receptor, immunopositive axons were distributed between odontoblasts. The ATP release by thermal stimulation from odontoblast-like cells was inhibited by the addition of siRNA for VNUT. These findings suggest that cytosolic ATP is transported by VNUT and that the ATP in the vesicles is then released from odontoblasts to ATP receptors on axons. ATP vesicle transport in odontoblasts seems to be a key mechanism for signal transduction from odontoblasts to axons in the pulp.

Kawasaki disease (KD) is a systemic vasculitis in infants that develops predominantly in the coronary arteries. Despite the clinically transient nature of active inflammation in childhood albeit rare complications (e.g., coronary artery aneurysm), KD has recently been suggested to increase the incidence of ischemic heart diseases in young adulthood. However, little is known about the histopathology of the coronary artery long after development of the acute KD vasculitis. To address this, we conducted histological studies of rabbit coronary arteries in adolescent phase after induction of the KD-like vasculitis induced by horse serum administration. After a transmural infiltration of inflammatory cells in acute phase at day 7, the artery exhibited a gradual decrease in the number of inflammatory cells and thickening of the intima during the chronic phase up to day 90, where proteoglycans were distinctly accumulated in the intima with abundant involvement of α-smooth muscle actin (α-SMA)-positive cells, most of which accompanied expression of VCAM-1 and NF-κB. Distinct from classical atherosclerosis, inflammatory cells, e.g., macrophages, were barely detected during the chronic phase. These observations indicate that the KD-like coronary arteritis is followed by intimal thickening via accumulation of proteoglycans and proliferation of α-SMA-positive cells, reflecting aberrant coronary artery remodeling.

The medullary vagal motor nuclei, the nucleus ambiguus (NA) and dorsal motor nucleus of the vagus (DMV), innervate the respiratory and gastrointestinal tracts. We conducted immunohistochemical analysis of expression of the androgen receptor (AR) and estrogen receptor α (ERα), in relation to innervation of the trachea and esophagus via vagal motor nuclei in mice. AR and ERα were expressed in the rostral NA and in part of the DMV. Tracing experiments using cholera toxin B subunit demonstrated that neurons of vagal motor nuclei that innervate the trachea and esophagus express AR and ERα. There was no difference in expression of sex steroid hormone receptors between trachea- and esophagus-innervating neurons. These results suggest that sex steroid hormones may act on vagal motor nuclei via their receptors, thereby regulating functions of the trachea and esophagus.

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